Dear Jacob, We have been using factor XA for several years for tag removal of the proteins we express in the facility. Factor XA does not leave an overhang, cutting after an IEGR sequence. It can be quite expensive though so we are looking into methods to purify it from bovine plasma.
David Blum Bioexpression and Fermentation Facility University of Georgia [email protected] bff.uga.edu On Mon, Apr 28, 2014 at 3:01 PM, Keller, Jacob <[email protected]>wrote: > Dear Crystallographers (this may be off-topic, depending on whom you > ask...) > > A: Can anyone recommend new improved alternatives to the usual suspects > for proteases (TEV, thrombin, enterokinase, etc.)? I've seen some > literature about SUMO- and NEDD8-dependent enzymes, but those apparently > require the whole protein domain to be in the construct for cleavage to > happen, rather than just a short sequence motif. Further, I'd be curious > whether there be drawbacks to using the various new breeds that might not > be mentioned in the original publications thereon. > > B: While I have seen several proteases that leave behind only 1 aa on the > new n-terminus of the target, I've yet to come across proteases that leave > behind very few residues on the new c-terminus, which would be very helpful > for tagging cleanly the c-terminus of proteins. Do such enzymes exist, and > if so, are there particularly good ones? And if not, I wonder why proteases > usually require more sequence on the n-terminal side of the scissile bond? > > All the best, > > Jacob Keller > > ******************************************* > Jacob Pearson Keller, PhD > Looger Lab/HHMI Janelia Farms Research Campus > 19700 Helix Dr, Ashburn, VA 20147 > email: [email protected] > ******************************************* >
